Date Published: October 1, 2018
Publisher: Public Library of Science
Author(s): Christopher N. Jondle, Kuldeep Gupta, Bibhuti B. Mishra, Jyotika Sharma, Dana J. Philpott.
Neutrophils are the first infiltrating cell type essential for combating pneumoseptic infections by bacterial pathogens including Klebsiella pneumoniae (KPn). Following an infection or injury, removal of apoptotic infiltrates via a highly regulated process called efferocytosis is required for restoration of homeostasis, but little is known regarding the effect of bacterial infection on this process. Here we demonstrate that KPn infection impedes the efferocytic uptake of neutrophils in-vitro and in-vivo in lungs by macrophages. This impaired efferocytosis of infected neutrophils coincides with drastic reduction in the neutrophil surface exposure of apoptosis signature phospholipid phosphatidyserine (PS); and increased activity of phospholipid transporter flippases, which maintain PS in the inner leaflet of plasma membrane. Concomitantly, pharmacological inhibition of flippase activity enhanced PS externalization and restored the efferocytosis of KPn infected neutrophils. We further show that KPn infection interferes with apoptosis activation and instead activates non-apoptotic programmed cell death via activation of necroptosis machinery in neutrophils. Accordingly, pharmacological inhibition of necroptosis by RIPK1 and RIPK3 inhibitors restored the efferocytic uptake of KPn infected neutrophils in-vitro. Importantly, treatment of KPn infected mice with necroptosis inhibitor improved the disease outcome in-vivo in preclinical mouse model of KPn pneumonia. To our knowledge, this is the first report of neutrophil efferocytosis impairment by KPn via modulation of cell death pathway, which may provide novel targets for therapeutic intervention of this infection.
Pneumonia is the most frequent cause of sepsis [1–3], which is one of the oldest and most elusive syndromes and a major challenge in medicine . With no effective therapies there are over 750,000 cases of sepsis each year in the United States alone, which accounts for 10% of all ICU patients, leading to a mortality rate between 20–50% depending on certain risk factors [5, 6]. In particular Klebsiella pneumoniae (KPn), an opportunistic pathogen, accounts for 5–20% of all Gram-negative sepsis cases [1, 3]. A notable emergence of antibiotic resistant strains of KPn in clinical settings has caused concerns over an already dwindling armamentarium of antibiotics. Thus, an understanding of host immune responses and pathogen-mediated manipulation thereof will likely provide novel therapeutic targets. In this regard, neutrophils are the first cell types to infiltrate the site of infection and contribute to the initial protective response. Indeed, in murine models of KPn infection, neutrophil-mediated responses are shown to be essential for initial control of the infection [7, 8]. We and others have shown that persistent accumulation of neutrophils and their over activation causes perpetuation of inflammation in pneumoseptic KPn infection [9, 10] [11–15]. Moreover, neutrophils have been reported to constitute a reservoir for this pathogen and aide in systemic dissemination of this infection . This underscores the importance of neutrophil turnover in KPn pneumonia and sepsis.
During an acute injury, neutrophils are promptly recruited to the site which, upon the resolution of insult, undergo apoptosis and are cleared by efferocytosis without inducing overt inflammation . Apoptotic cells are swiftly removed by efferocytic cells by recognition of surface “eat me” signal phosphatidylserine (PS), exposed as a result of caspase-mediated inhibition of flippase activity on apoptotic cell membrane . Here we report that Klebsiella pneumoniae, an opportunistic pathogen, prevents efferocytic clearance of neutrophils by reducing surface exposure of PS via modulation of flippase activity. We also show that KPn infection skews the programmed cells death away from “efferocytosis favorable” apoptosis toward necroptosis, which is likely the associated with increased neutrophilia and poor disease outcome. Concomitantly, reversal of PS exposure and blockage of necroptosis improves efferocytic clearance of neutrophils as well as disease outcome in murine inhalation model of KPn pneumonia. Our study provides important insights into pathogenic mechanisms that can be targeted for future antimicrobial therapies for this infection.